Stimulated secretion of granules from cultured RBL-2H3 mast cells is dependent on mobilization of calcium via phospholipase (PL) C and activation of protein kinase (PK) C by diglycerides generated through PLC and PLD. In recent years we have focussed on PLD because this enzyme is the primary source of diglycerides in stimulated mast cells and its activation is essential for secretion (see previous reports in this series). In intact cells. PLD is activated by antigen and synergistically by stimulants of CaM kinase II (thapsigargin), PKC (phorbol ester) and PKA (cholera toxin). The activation of PLD by antigen is partially blocked by inhibitors of each of these kinases and is totally blocked by inhibitors of all three kinases in combination. The synergistic regulation of PLD by CaM kinase II. PKC, and PKA was also apparent from studies in permeabealized RBL-2H3 cells. These studies also showed that activation of PLD, along with an increase in cytosolic calcium ions, was a necessary signal for secretion. For example, in all experiments the extent of activation of PLD and secretion were highly correlated but only when when stimulation was accompanied by a calcium signal. In addition, secretion was suppressed by primary alcohols (but not by secondary alcohols)that divert production of the PLD product, phosphatidic acid to phosphatidyl alcohol (the so called transphosphatidylation reaction). RBL-2H3 cells were found to express message and protein for the two known forms of PLD, namely PLD1 and 2. Accordingly, we have investigated the distribution and function of these two isoforms by subcellular fractionation techniques and expression of wild type and catalytically inactive mutants of PLD1 and 2 fused with green fluorescent protein. These experiments indicated 1) PLD1 and PLD2 were located on secretory granules and plasmam membrane, respectively, 2)both isoforms were activated by antigen and synergistically by stimulants of CaM kinase II, PKC, and PKA, 3) after stimulation of cells PLD1 migrates along with granules to the cell periphery and fuses with the plasma membrane, and 4) expression of the mutated inactive forms of PLD1 and 2 blocks migration of granules and their fusion with the plasma membrane. The studies to date have thus demonstrated that both PLD1 and 2 respond to secretory stimuli and participate in the secretory process; PLD1 in the translocation of granules and PLD2, possibly in conjunction with PLD1, in the final fusion events. We are now investigating the mechanisms by which the protein kinases regulate the PLDs and future work will focus on the mechanisms by which PLD regulate secretion. Some of the above findings have been verified in human mast cells derived from peripheral blood stem cells. The human mast cells, unlike RBL-2H3 cells,have allowed comparison of signaling processes including activation of PLD that are induced by growth factors as well as antigen (see also accomapnying report Z01 HL00990-14).